Despite the clinical importance of embryonic stem (ES) cells for regenerative medicine, many key biological features of these cells remain unexplored. We recently discovered that core components of the apoptotic machinery are differentially regulated in ES cells and that this unique regulation is critical for the ability ofES to respond rapidly to DNA damage. In particular, we found that undifferentiated human (h) ES cells are primed for rapid apoptosis by maintaining the pro-apoptotic protein Bax in its active conformation at the Golgi. Remarkably, we found that just two days of differentiation induced significant changes: Bax was no longer in an active state and the cells were no longer highly sensitive to DNA damage. Thus, the apoptotic machinery undergoes extraordinary changes even in the earliest stages of hES cell differentiation. Rather unexpectedly, I found that changes in these apoptotic components affected not only the thresholds of apoptosis but also the ability of ES cell to maintain self-renewal. Specifically, I found that levels of the anti- apoptotic proten Mcl-1 are markedly reduced with early differentiation and that inhibiting Mcl-1 alone was sufficient to induce differentiation of hES cells. These results have lead me to hypothesize that apoptotic proteins that were previously thought to be involved only in processes of cell death, are molecular switches controlling stem cell fate. My overall plan is to investigate the molecular intersection between apoptosis and stem cell differentiation with the goal of determining whether these pathways are hijacked by cancer stem cells for the dual purpose of resisting apoptosis and maintaining self renewal. Specifically, in Aim 1 I will examine the pathways that regulate Bax function during ES cell differentiation. My results also show that reprogramming of fibroblasts into induced pluripotent stem (iPS) cells is coupled with increased sensitivity to apoptosis. I will examine how the pluripotency transcription factors affect apoptosis thresholds. In Aim 2, I will focus my studies on Mcl-1 to determine the mechanism by which Mcl-1 regulates self-renewal in ES cells. In Aim 3, I will use a model of glioblastoma cancer stem cells to determine whether inhibition of Mcl-1 activity can be used as therapeutic tool to simultaneously increase the sensitivity to apoptosis and restrict the self-renewal capability. These studies will undoubtedly uncover critical aspects of apoptosis regulation in ES cells and reveal key features of stem cell biology that can have significant impact for cancer therapy.